PROJECT SUMMARY The tightly regulated balance between proliferation and differentiation of basal stem/progenitor cells of the oral epithelium is critical for proper tissue development, repair, renewal, and to maintain homeostasis. Therefore, the development of new tools and strategies directed at identifying transcriptional and signaling networks underlying stem/progenitor cell function of the oral epithelium are critical. Hence, our goal is to examine the molecular mechanisms of the transcriptional and gene-regulatory mechanisms that control stem/progenitor cell function of the oral epithelium with the ultimate goal for using the knowledge gained from such studies towards stem cell regenerative-based therapies and tissue engineering approaches. It is well established that ?Np63 plays a critical role in epithelial regenerative function as ?Np63-null animals fail to develop several epithelial-rich organs including those of the oral cavity. However, our current knowledge of how ?Np63 interacts with and shapes the chromatin and transcriptional regulatory environment of the stem/progenitor cells of the oral epithelium, is lacking. To address these knowledge gaps, we will utilize an enriched population of oral epithelial stem/progenitor cells obtained from novel ?Np63-GFP transgenic mice to study two major areas of interest. First, we will perform both clonogenic and functional assays to compare the abilities of ?Np63-GFPhi, ?Np63-GFPlow, ?Np63neg and ?Np63-GFPhi-KD (?Np63 specific inducible knockdown in ?Np63-GFPhi cells using siRNA mediated strategies) oral epithelial cells to retain their progenitor capabilities in organospheres (Aim1A). Furthermore, we will perform transcriptomic profiling (RNA-seq) to generate global gene expression profiles of ?Np63-GFPhi, ?Np63-GFPlow, ?Np63neg and ?Np63-GFPhi-KD to better understand the ?Np63-dependent gene regulatory mechanisms that are important for oral epithelial stem/progenitor cell biology (Aim1B). Such studies are important, since they will identify for the first time the gene expression profile of oral epithelial stem/progenitor cells on a broad and dynamic scale. Second, to examine the global status of the chromatin architecture of oral epithelia cells, we will perform ATAC-seq experiments with ?Np63- GFPhi, ?Np63-GFPlow, ?Np63neg and ?Np63-GFPhi-KD cells to identify the ?Np63 dependent and independent regulatory chromatin environment that are important for stem/progenitor cell function (Aim 2). Collectively, our approach using a genetically-defined model system and cutting-edge next generation sequencing technology will better elucidate the transcriptomic and epigenomic landscape of oral stem/progenitor cells and shed light on the ?Np63-governed transcriptional regulatory network and signaling pathways. This work is highly innovative and significant because our proposed use of sophisticated genetic tools, in vivo models and genome-wide profiling assays to examine fundamental transcriptional control mechanisms will lead to new discoveries important for oral epithelial stem cell based regenerative strategies used to treat and regenerate oral tissues following injury, damage or in diseased states.